Kean Lewis opened the scoring for Mohun Bagan in the 13th minute with a fine header, but the Sri Lankan hosts roared back courtesy of Afeez Olofin's equalising goal 30 minutes into the game.
Federation Cup champions Mohun Bagan bagged the winner in the 70th minute with Sehnaj Singh finding the net to secure a vital one-goal advantage for the Kolkata-based visitors.
The winner of the preliminary round will feature in another double-legged play-off match before qualifying for the main AFC Cup, the continent's second tier club competition.
Mohun Bagan coach Sanjoy Sen made a few changes with Shilton Paul starting at the goal and Prabir Das and Kean Lewis on either wings. Sehnaj Singh partnered Souvik Chakraborti in midfield while Daryl Duffy was the solitary striker upfront with Katsumi Yusa operating behind.
The Kolkata side looked dangerous in the opening minutes with Yusa inches away from a Prabir Das cross and Pritam Kotal finding himself in plenty of space in the opposition box after a hopeful long ball, only to be given off-side.
Prabir, in turn, sent the ball towards the far post and Lewis met it with a diving header, giving Colombo goalkeeper Mohammed Imran no chance.
In the 20th minute, Yusa had another chance to find the net but Imran got a fingertip to it after Pritam had found him with a perfect delivery from the right.
Colombo had a clutch of chances with Dilan and Afeez wreaking havoc in the Bagan box. Opayemi was unlucky to see his header go wide off a corner before Afeez finally struck at the half hour mark.
Five minutes later, Prabir was guilty of heading wide from an unmarked position inside the box after Kotal had put him on the clear. Both sides were locked 1-1 at half time.
After resumption, Bagan seemed to grow in confidence but misses from Duffy and Jeje did not help their cause.
Mohun Bagan finally had the winner after 70 minutes following a belter from Sehnaj that gave Colombo goalkeeper Imran no chance. This was after Shouvik Ghosh had sent in a cross into the box that was left by Jeje for the onrushing Sehnaj who hit it first time with a lot of power.
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